CATRINA (2015), 12 (1): 31-36
© 2015 BY THE EGYPTIAN SOCIETY FOR ENVIRONMENTAL SCIENCES
Molecular and pharmacological investigation of Stipagrostis scoparia growing in North Sinai
Mayada M. El Ayouty1, Hashim A. Hassanean2, Rawia Zayed1, Mostafa K. Mesbah1 and Amany K. Ibrahim2*
1
Department of Pharmacognosy, Faculty of Pharmacy, Sinai University, AlArish, North Sinai, Egypt
2
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, 41522, Ismailia, Egypt
ABSTRACT
Although many plants of Poaceae family attracted several investigators regarding their pharmacological
aspects, there is almost no report on the pharmacology of Stipagrostis scoparia. Accordingly, it was
necessary to carry out pharmacological study on the wild Egyptian plant which grows in North Sinai and
selectively discriminate the plant using Random Amplified Polymorohic DNA technique. RAPD DNA
analysis of the plant was performed using ten decamer primers. It showed distinguishable bands and
generated 72 fragment patterns. The analysis of RAPD data can select the use of primers Z-02, C-17 and
Z-08 for the selective discrimination of Stipagrostis scoparia growing in Egypt. Concerning the
biological studies, the methanolic extract of Stipagostis scoparia exhibited significant analgesic, antiinflammatory and anti-oxidant activities after in-vivo assays. Results obtained on assessing the long-term
anti-hyperglycemic effect revealed a significant reduction in blood glucose level in Alloxan-diabetic rats
treated with the methanolic extract. Prolonged administration of the methanolic extract significantly
decreased the level of liver enzymes in carbon tetrachloride-intoxicated rats. According to the acute
toxicity studies it was found that the methanolic extract of Stipagrostis scoparia is safe up to 7 g/kg b.wt.
Key words: Stipagrostis scoparia; RAPD analysis; analgesic; anti-inflammatory; anti-oxidant.
INTRODUCTION
The genus Stipagrostis contains species that occur in
xerophytic open habitats that include desert and semidesert and also dunes. There are 50 species occurring in
Africa, Southwest Asia and Northwest India.
Stipagrostis scoparia (Trin. & Rupr.) de Winter is a
North African perennial grass which appears to be
dominant in North Sinai dune-fields; it is used among
Bedouins for grazing purposes of their camels (Arab
Millennium Ecosystem Assessment, 2006; Raafat et al.,
2008). For selective discrimination of Stipagrostis
scoparia growing in Egypt Random Amplified
Polymorohic DNA (RAPD) analysis was performed.
Plant genotype, or DNA fingerprinting of plants is a
technology that has matured and is poised for very
widespread practical application. Plant genotype
analysis has application in the identification of plants in
commerce, plant breeding and research. Commercial
applications include the protection of plant breeder’s
rights and patents, quality control in plant production
and processing and labeling of plant-derived foods and
other products (Henry, 2001).
The simplicity and applicability of the RAPD
technique have captivated many scientists’ interests.
Perhaps the main reason for the success of RAPD
analysis is the gain of a large number of genetic markers
that require small amounts of DNA without the
requirement for cloning, sequencing or any other form
of the molecular characterization of the genome of the
species in question (Bardakci, 2001).
The grasses of Poaceae are “ecologically the most
dominant and economically by far the most important
family in the world” (Heywood, 1978). Some of the
grass species have been proved to show strong
antioxidant properties and have been effective in the
treatment of inflammations (Rice-Evans et al., 1996;
Adom and Liu, 2002; Jana et al., 2009). The fresh or
dried rhizomes of P. communis (Rhizoma Phragmites)
are used in diabetic complications in the traditional
____________________________________________
* Corresponding author: am_kamal66@yahoo.com
Chinese Medicine (TCM) (Li et al., 2004). The herb is
also used to treat breast cancer, leukaemia and diabetes
in TCM (Rice-Evans et al., 1996). There is almost no
report on the pharmacology of Stipagrostis scoparia.
Accordingly, it was necessary to carry out
pharmacological study on the wild Egyptian plant which
grows in North Sinai.
MATERIALS AND METHODS
Materials for DNA profiling
Extraction buffer n: 2% N-cetyl-N, N, N-trimethyl
ammonium bromide (CTAB), 0.1 M Tris-HCI
(Hydroxyl methyl amino methane with HCl to pH 8),
0.02 M EDTA, 1.4 M NaCl, 1% (v/v) mercaptoethanol (added immediately before use).
Washing buffer 1: 76% ethanol, 0.2 M Na-acetate.
Washing buffer 2: 76% ethanol, 10 mM NH4-acetate.
TE-buffer: 10 mM Tris-HCl (pH=8.0), 1 mM EDTA.
10X incubation buffer: 100 mM Tris-HCl (pH 8.3), 500
mM KCl, 0.01% (w/v) gelatin. Taq DNA polymerase
(Perkin-Elmer/Cetus, USA; Advanced Biotechnologies,
UK). RNAse: Boehringer Mannheim. DNTP's mix:
Pharmacia, Sweeden. Ten primers: purchased from
Operon Technologies Inc. (Almeda, California, USA),
were used for Random Amplified Polymorphic DNA
(RAPD) analysis, with the following sequences: D-01
5'-ACCGCGAAGG-3', C-17 5'-TTCCCCCCAG-3', M03 5'-GGGGGATGAG-3', M-16 5'-GTAACCAGCC-3',
O-09 5'-TCCCACGCAA-3', B-16 5'-TTTGCCCGGA3',
Z-02
5'-CCTACGGGGA-3',
Z-04
5'AGGCTGTGCT-3', Z-08 5'-GGGTGGGTAA-3' and Z13 5'-GACTAAGCCC-3'.
Materials for evaluation of the biological effects
Experimental models
Albino mice (20-25 g), used for determination of
LD50 and analgesic activity; adult male albino rats of
Sprague Dawley Strain (130-150 g), utilized for
assessment of the different pharmacological effects,
Molecular and pharmacological investigation of Stipagrostis scoparia
annealing step at 36ºC for 1 min, and an elongation step
at 72ºC for 1.5 min. The primer extension segment was
extended to 7 min at 72ºC in the final cycle.
Electrophoresis and visualization of PCR
product
The amplification products were resolved by
electrophoresis in a 1.5 % agarose gel containing
ethidium bromide (0.5 µg/ml). PCR products were
visualized on UV light and photographed using a
Polaroid camera.
were obtained from the animal house colony at the
National Research Center (Dokki, Giza, Egypt).
Drugs and kits
Alloxan: (Sigma, USA) solution (10 mg/0.1 ml).
Carrageenan: (Sigma, USA). Indomethacin: Egyptian
International Pharmaceutical Industries Co, (EIPICO,
under license of Merck & Co. INC-RAHAWY N.J.,
USA). Silymarin: Sedico Pharmaceutical Co., 6 October
City, Egypt. Vitamin E: (dl-α-tocopheryl acetate):
Pharco Pharmaceutical Co. Metformin: (Cidophage®):
Chemical Industries Development Co. (CID Co.), Giza,
Egypt. Biomerieux kit: used for the assessment of blood
glucose level. Biodiagnostic kit: Wak-Chemie Medical
Germany (for measuring the antioxidant activity).
Transaminase Kits (Bio-Merieux Co., France)
biochemical kits for assessment of serum levels of
alanine
aminotransferase
(ALT),
aspartate
aminotransferase (AST) and alkaline phosphatase
(ALP) enzymes.
Biological studies
Acute toxicity studies (Determination of median
lethal dose, LD50)
Determination of the LD50 of the methanolic extract
of S. scoparia was estimated according to the Karber
method, (1931). Preliminary experiments were done to
determine the minimal dose that kills all animals
(LD100) and the maximal dose that fails to kill any
animal. Several doses at equal logarithmic intervals
were chosen in between these two doses, each dose was
injected in a group of six animals by subcutaneous
injection. The mice were then observed for 24 hours and
symptoms of toxicity and mortality rates in each group
were recorded and the LD50 was calculated.
Using the carrageenan-induced rat paw oedema test
as described by Winter et al., (1962), Twenty-four male
albino animals divided in to four groups (each of six
animals) were used. They were administered one single
oral dose of the tested extract at a dose of (100 mg/kg
b.wt.) and the reference drug, Indomethacin at a dose of
(20 mg/Kg. b.wt.). The negative control group received
saline. One hour later all the animals had a subplanter
injection of 0.1 ml of 1% carrageenan solution in saline,
in the right hind paw and 0.1% of saline in the left hind
paw. Four hours after drugs administration, the rats
were sacrificed. Both hind paws were excised and
weighed separately.
The percentage of oedema produced and that of
oedema inhibition due to drug administration were,
respectively calculated as follows:
Plant material
The plant was collected from El Arish and kindly
authenticated by Prof. Dr. Abd Elraof A. Professor of
Botany, Faculty of Science, Suez Canal University. A
voucher specimen (# SAA-153) was deposited in the
herbarium section of Pharmacognosy Department,
Faculty of Pharmacy, Suez Canal University, Egypt. For
DNA profiling, the whole fresh plant was collected
during the year 2010, freeze-dried and ground to fine
powder under liquid nitrogen. For biological screening,
the extract was prepared after extraction of the whole
plant by maceration with 90% methanol till complete
exhaustion, filtered, evaporated till dryness.
DNA fingerprintig of Stipagrostis scoparia
DNA extraction
The plant powder was mixed with buffer n and
incubated for 10 min at 65ºC. It was mixed about 2-3
times during incubation by inverting tube. The mixture
was extracted with chloroform: isoamylalcohol (24: 1)
and centrifuged at 10.000 rpm for 10 min. The DNA in
the aqueous phase was precipitated by the addition of 3
M sodium acetate and isopropanol. The precipitates
were washed in 70% ethanol and dissolved in a small
volume of TE buffer. Polymerase Chain Reaction (PCR)
amplifications were tested with RAPD primers from
commercially available kits D-01, C-17, M-03, M-16,
O-09, Z-02, Z-04, Z-08, Z-13, and B-16. The
amplification reaction was carried out in 25 μl reaction
volume containing 10X incubation buffer, 1.5 mM
MgCl2, 0.2 mM dNTPs, 1 μM primer F, 1 μM primer R,
1 U Taq DNA polymerase and 25ng template DNA.
Thermocyling Profile and Detection of the PCR
Products
PCR amplification was performed in a PerkinElmer/GeneAmp® PCR System 9700 (PE Applied
Biosystems) programmed to fulfill 40 cycles after an
initial denaturation cycle for 5 min at 94ºC. Each cycle
consisted of a denaturation step at 94ºC for 1 min, an
Oedema = (Wt. of right paw - Wt. of left paw)
×100/Wt. of left paw
% Oedema inhibition = (Mc – Mt) × 100/Mc
Where, Mc is the mean oedema in control rats and Mt is the
mean oedema in drug-treated animals.
Analgesic activity
Swiss male albino mice (20-25 g) were used.
Animals were acclimatized to the laboratory conditions
for at least one hour before testing. The analgesic
activity was estimated using acetic acid induced
writhing test, the extract was administrated orally at a
dose of (100 mg/kg b.wt.) and the reference drug,
indomethacin at a dose of (20 mg/Kg. b.wt.). Thirty
minutes later 0.6% acetic acid was injected
intraperitonial (0.2% ml / mouse) each mouse was then
placed in an individual clear plastic observation
32
Ibrahim et al.
chamber and the total number of writhes/minute was
counted for each mouse (koster et al., 1959).
Antioxidant activity
Diabetes mellitus was induced to male albino rats of
the Sprague Dawely Strain (130-140 g) by
intraperitoneal injection of alloxan (150 mg/kg b.wt.) as
described by (Eliasson and Samet, 1969).
Hyperglycaemia was assessed after 72 hours by
measuring blood glucose (Trinder, 1969) using 36
diabetic rats divided into 6 groups each of 6 animals.
They were administered one single dose of the tested
samples and the reference drug in specific doses but the
negative control and positive control groups received
saline. Blood samples were collected after 7 days for
estimation of blood glutathione level using
biodiagnostic glutathione kit for assessment of
antioxidant activity. The restoration of blood
glutathione levels (reduced due to induction of diabetes)
was taken as a measure of antioxidant activity. The
percentage change observed after dose administration
was, in each case, calculated according to the following
equation:
iasson and Samet, 1969). Hyperglycemia was assessed
after 72 hours by measuring blood glucose (Trinder,
1969) and after 2 weeks and 4 weeks intervals. They
were administered one single dose of the tested samples
and the reference drug in specific doses but the negative
control and positive control groups received saline. At
the end of each study period, blood samples were
collected from the retro-orbital venous plexus through
the eye canthus of aneasthetized rats after an overnight
fast. Serum was isolated by centrifugation and the blood
glucose level was measured (Trinder, 1969).
The percentage of change in blood glucose level
from initial glycemia was, in each case, calculated
according to the following equation:
% of change = (G0–Gt) × 100/G0
RESULTS
DNA fingerprintig of Stipagrostis scoparia
The banding profile produced by the ten decamer
primers used in RAPD analysis of Stipagrostis scoparia
which grows in North Sinai of Egypt is illustrated in fig.
(1). The RAPD electrophoretic profile of DNA sample
amplified with the ten decamer primers showed
distinguishable bands and generated 72 fragment
patterns. RAPD fragments showed 10 bands by Z-02, 9
bands by C-17 and Z-08, 8 bands by Z-04, 7 bands by
D-01 and M-16, 6 bands by Z-13 and B-16 and 5 bands
by M-03 and O-09. The analysis of RAPD data can thus
select the use of primers Z-02 (10 bands) and C-17 and
Z-08 (9 bands) for the selective discrimination of
Stipagrostis scoparia growing in Egypt.
% of change = (G–G0) ×100/G0.
Where, G0 represents the glutathione level in diabetic animals,
prior administration of the samples and G that measured after.
Hepatoprotective activity
Liver damage in rats was induced according to the
method of (Klassen and plaa, 1969) by intraperitoneal
injection of 5 ml/kg of 25 % carbon tetrachloride (CCl 4)
in liquid paraffin. Seventy-two hours after
administration of CCl4, blood samples were withdrawn
to be used for the biochemical study.
Adult male albino rats of Sprange Dawely Strains
(130-140 g) were randomly divided into 5 groups each
of 10 animals. The methanolic extract of the plant (100
mg/kg b.wt.), as well as, the standard drug (Silymarin,
25 mg/kg b.wt.), were separately administered daily for
one week before and one week after liver damage. A
group of animals were kept untreated (receiving only
saline) and served as a negative control.
Biochemical studies were carried out; followed by
an overnight fast; whole blood was obtained from the
retro-orbital venous plexus through the eye canthus of
anaesthetized rats. The blood samples were collected at
zero time, after one week of receiving the tested drug,
72 hours after induction of liver damage then after a
week of treatment with the tested samples and standard.
Serum was isolated by centrifugation and divided for
analysis of aspartate amino-transferase (AST), alanine
amino-transferase (ALT) (Thefweld et al., 1974) and
alkaline phosphatase (ALP) enzymes (Kind and King,
1954).
Hypoglycemic activity
Male albino rats of the Sprague Dawely Strain (130140 g) were injected intra-peritoneal with alloxan (150
mg/kg body weight) to induce diabetes mellitus (Eli-
Figure (1): The obtained RAPD-PCR product for Stipagrostis
scoparia using ten decamer primers.
Biological studies
Acute toxicity studies (Determination of median
lethal doses, LD50)
It was found that the methanolic extract of
Stipagrostis scoparia is safe up to 7 g/kg b.wt.
Anti-inflammatory activity
The anti-inflammatory activity (table 1) of the
extract of Stipagrostis scoparia was evaluated on
carrageenan-induced rat hind paw oedema model. The
extract (100 mg/kg) has been found to possess
significant anti-inflammatory activity on the tested
experimental model.
33
Molecular and pharmacological investigation of Stipagrostis scoparia
Table (1): acute anti-inflammatory activity of the methanolic extract of Stipagrostis scoparia plant and indomethacin drug in male
albino rats (n=6).
Group
Dose in
mg/kg.b.wt
Control
S. scoparia
Indomethacin
I ml Saline
100
20
% Oedema
Mea± S.E.
% of Change
60.3±1.7
ــــــ
32.4±1.1*
46.3
21.8±0.6*
63.8
Potency1
ــــــ
ــــــ
0.73
1
1
Potency calculated as compared to the standard anti-inflammatory drug indomethacin,*Significantly different from control group at P<0.01,
S.E. = Standard Error, % of change is calculated as regards to the control group.
Analgesic activity
The analgesic activity (table 2) of the extract of Stipagrostis scoparia was estimated using acetic acid induced
writhing test. The extract (100 mg/kg) has been found to possess significant analgesic activity on the tested
experimental model.
Table (2): Effect of methanolic extract of Stipagrostis scoparia plant on number of abdominal constrictions and indomethacin in
mice (n=6).
Group
Dose mg/kg b.wt. Number of abd. constrictions % inhibition Potency1
Control
1 ml saline
47.2±1.4
ــــــــ
ـــــــ
S. scoparia
100
29.3±0.7*
37.9
0.63
Indomethacin
20
18.7±0.4*
60.4
1
1
Potency calculated as compared to the standard anti-analgesic drug Indomethacin, *Significantly different from control group at P<0.01, S.E. =
Standard Error,,% of inhibition is calculated as regards to the control group.
Antioxidant activity
The antioxidant activity of the extract of Stipagrostis scoparia was estimated using the restoration of blood
glutathione levels (reduced due to induction of diabetes) as a measure of antioxidant activity. The extract (100 mg/kg)
has been found to possess significant antioxidant activity on the tested experimental model. Results obtained for the
methanolic extract are recorded in (table 3)
Table (3): Antioxidant activity of the methanolic extract of Stipagrostis scoparia plant and vitamin E drug in male albino rats (n=6).
Group
Blood glutathione (mg %)
% change from control
Potency1
Control (1 ml saline)
Daibetic
Diabetic + Vitamin E (7.5m/kg)
36.4±1.2
22.1±0.4*
35.8±1.1
ـــــــــــــــ
ـــــــــــــــــــ
61.9
ـــــــــــــــــــ
ـــــــــــــــــــ
1
Diabetic + extract (100mg/kg)
35.3±0.9
59.7
0.96
1
Potency calculated as compared to the standard anti-oxidant drug vitamin E, * Statistically significant different from negative control group at p <
0.10, % of inhibition is calculated as regards to the positive control group.
Hepatoprotective activity
The data obtained were analyzed using student's t- test where means of the treated groups were compared to that of
the control group for each variable. Results obtained for the methanolic extract are recorded in (table 4)
Table (4): Effect of the methanolic extract (100 mg/kg) of Stipagrostis scoparia plant and silymarin drug on serum enzymes level
(AST, ALT and ALP) in liver damaged rats (n=6).
Group
AST (u/L)
Control
Zero
41.8±1.3
72h
131.8±4.6*
7d
161.4±0.9*
ALT (u/L)
Zero
36.8±1.2
72h
141.4±4.2*
ALP (KAU)
extract
43.2±1.7
81.7±1.8*
63.8±1.9*
33.5±1.1
76.2±2.1*
Silymarin
38.6±1.4
56.4±1.9*
34.9±1.1
39.1±1.3
51.7±1.4*
7d
156.8±
0.9*
59.1±
2.1*
38.2±
1.1
* Statistically significant from zero time at p < 0.01,.Statistically significant from 72h after ccl4 at p <0.01.
34
Zero
7.1±
0.1
7.3±
0.1
7.2±
0.1
72h
41.6±
1.8*
29.8±
0.9*
19.3±
0.4*
7d
56.7±
2.1*
24.3±0.
5*
7.5±
0.1
Ibrahim et al.
constituents of antioxidants as the antioxidant
compounds have been reported to beneficially improve
pancreatic β-cell function by preventing or delaying βcell dysfunction due to glucose toxicity as in the
aqueous extract of the leaves of Bambusa arundinacea
(Joshi et al., 2009) and improve the liver function as in
the methanolic extract of Hordeum vulgare Linn.
(Poaceae) seeds (Shah et al., 2009). Flavonoids which
are some of the constituents of Poaceae were found to
have anti inflammatory properties (Trease and Evans,
1989; Parmer and Gosh, 1978). It contributed to the anti
inflammatory activity of the ethanolic extract of Setaria
megaphylla (Poaceae) Leave (Okokon, et al., 2006) so
the flavonoidal content of Stipagrostis scoparia may be
the main cause of the anti-inflammatory activity
exhibited by the methanolic extract.
The analgesic activity may be contributed to the
content of the essential oil as in Cymbopogon
winterianus (Poaceae) (Quintans-Ju´ nior et al.,
2008).This study opens the gate towards mining for the
secondary metabolites responsible for the mentioned
interesting activities within the tissues of Stipagrostis
scoparia.
Hypoglycemic activity
A strong significant hypoglycimic effect in alloxan
induced diabetes mellitus rats was observed for the
methanolic extract in a dose of 100 mg/kg. The results
are listed in table (5). Nuclear DNA markers usage was
a useful tool to explore the origin, diversity, and
parentage of many plants as in Spartina anglica
(Poaceae), Such primers are used mainly to discriminate
the plant (Ayres and Strong, 2001). In this study
Stipagrostis scoparia was subjected to RAPD assay;
this was performed using ten different primers and it
can be concluded that the most relevant fragment
resulting from the successful combination of template
and primer was that produced by Z-02, C-17 and Z-08
RAPD primers.
Secondary metabolites as phenolic compounds and
flavonoids commonly occur in both cultivated and wild
Poaceae (Sánchez–Moreiras et al., 2004) and this
content of the phenolic compounds may be the cause of
its antioxidant activities as in Saccharum officinarum
(Poaceae) leave extracts (Ghiware et al., 2012). The
hypoglycemic and the hepatoprotective activities of
Stipagrostis scoparia could be attributed to its
Table (5): Effect of the methanolic extract of S. scoparia plant and metformin drug on blood glucose level in male albino rats.
Group
Time
Zero
2 weeks
4 weeks
Diab.
non
treated
Mean±S.E
251.6±6.4
262.3±6.9
265.2±5.7
Diab. treated with methanolic
extract (100 mg/kg)
M±S.E
% of change
263.8±9.2
186.4±6.3*
28.9
132.3±4.7*
50.1
Diab. treated with metformin (100
mg/kg)
M± S.E
% of change
265.7±8.9
136.2±4.2*
48.1
85.6±2.3*
67.7
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